Human pancreatic islets were isolated from a cadaveric donor. The population was 70% pure in mature islets, which was determined by dithizone staining. Islets cells were expanded according to Pharmaceutical Production Research Facility Protocol (PPRF) for 6 weeks. Expansion phase: Approximately 2,500 islet equivalents were seeded onto 75-cm2 fibronectin-coated tissue culture-treated flasks in a proprietary serum-free medium (PPRF medium). The serum-free PPRF medium was supplemented with 30% serum-free mesenchymal-conditioned medium. When the cell migration resulted in the formation of large-size colonies, the cells were harvested by trypsinization and replated into new tissue culture flasks. Once the cells reached near confluence (80–90%) in monolayer, they were trypsinized, counted, and subcultured at a density of 5,000 cells per cm2. Thereafter, cells were serially passaged using the same protocol.

Human pancreatic islets were isolated from a cadaveric donor. The population was 70% pure in mature islets, which was determined by dithizone staining. Islets cells were expanded for 6 weeks and re-differentiated for 2 days according to Pharmaceutical Production Research Facility (PPRF) Protocol. Re-differentiation phase: For induction of expanded cells to differentiate into islet-like cell clusters, the cells were seeded into 60 mm ultra-low attachment dishes in serum-free CMRL-1066 supplemented with 4 mM L-glutamine, 1% BSA, insulin (10 g/ml), transferrin (5.5 g/ml), sodium selenite (6.7ng/ml), and 1% antibiotic antimycotic solution at a density of 6.0 x 10EXP6 cells per dish. The induction medium was further added with islet neogenesis-associated protein (INGAP) peptide at a concentration of 1.0ug/ml.